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1.
Exp. mol. med ; Exp. mol. med;: e351-2017.
Article in English | WPRIM | ID: wpr-153368

ABSTRACT

NHERF1/EBP50 (Na⁺/H⁺ exchanger regulating factor 1; Ezrin-binding phosphoprotein of 50 kDa) organizes stable protein complexes beneath the apical membrane of polar epithelial cells. By contrast, in cancer cells without any fixed polarity, NHERF1 often localizes in the cytoplasm. The regulation of cytoplasmic NHERF1 and its role in cancer progression remain unclear. In this study, we found that, upon lysophosphatidic acid (LPA) stimulation, cytoplasmic NHERF1 rapidly translocated to the plasma membrane, and subsequently to cortical protrusion structures, of ovarian cancer cells. This movement depended on direct binding of NHERF1 to C-terminally phosphorylated ERM proteins (cpERMs). Moreover, NHERF1 depletion downregulated cpERMs and further impaired cpERM-dependent remodeling of the cell cortex, suggesting reciprocal regulation between these proteins. The LPA-induced protein complex was highly enriched in migratory pseudopodia, whose formation was impaired by overexpression of NHERF1 truncation mutants. Consistent with this, NHERF1 depletion in various types of cancer cells abolished chemotactic cell migration toward a LPA gradient. Taken together, our findings suggest that the high dynamics of cytosolic NHERF1 provide cancer cells with a means of controlling chemotactic migration. This capacity is likely to be essential for ovarian cancer progression in tumor microenvironments containing LPA.


Subject(s)
Cell Membrane , Cell Movement , Cytoplasm , Cytosol , Epithelial Cells , Membranes , Ovarian Neoplasms , Pseudopodia , Tumor Microenvironment
2.
Exp. mol. med ; Exp. mol. med;: e309-2017.
Article in English | WPRIM | ID: wpr-198940

ABSTRACT

Hepatocyte growth factor (HGF) and its receptor, cMET, play critical roles in cell proliferation, angiogenesis and invasion in a wide variety of cancers. We therefore examined the anti-tumor activity of the humanized monoclonal anti-HGF antibody, YYB-101, in nude mice bearing human glioblastoma xenografts as a single agent or in combination with temozolomide. HGF neutralization, The extracellular signal-related kinases 1 and 2 (ERK1/2) phosphorylation, and HGF-induced scattering were assessed in HGF-expressing cell lines treated with YYB-101. To support clinical development, we also evaluated the preclinical pharmacokinetics and toxicokinetics in cynomolgus monkeys, and human and cynomolgus monkey tissue was stained with YYB-101 to test tissue cross-reactivity. We found that YYB-101 inhibited cMET activation in vitro and suppressed tumor growth in the orthotopic mouse model of human glioblastoma. Combination treatment with YYB-101 and temozolomide decreased tumor growth and increased overall survival compared with the effects of either agent alone. Five cancer-related genes (TMEM119, FST, RSPO3, ROS1 and NBL1) were overexpressed in YYB-101-treated mice that showed tumor regrowth. In the tissue cross-reactivity assay, critical cross-reactivity was not observed. The terminal elimination half-life was 21.7 days. Taken together, the in vitro and in vivo data demonstrated the anti-tumor efficacy of YYB-101, which appeared to be mediated by blocking the HGF/cMET interaction. The preclinical pharmacokinetics, toxicokinetics and tissue cross-reactivity data support the clinical development of YYB-101 for advanced cancer.


Subject(s)
Animals , Humans , Mice , Antibodies, Neutralizing , Cell Line , Cell Proliferation , Glioblastoma , Half-Life , Hepatocyte Growth Factor , Heterografts , In Vitro Techniques , Macaca fascicularis , Mice, Nude , Pharmacokinetics , Phosphorylation , Phosphotransferases , Toxicokinetics
3.
Article in English | WPRIM | ID: wpr-106242

ABSTRACT

PURPOSE: Hollow fiber assays offer an early in vivo method of anticancer drug screening. The assays have been optimized for human cancers originating from the lung, breast, colon, ovary, and brain, but not from the stomach and liver. The current study focused on optimization of hollow fiber assays for gastric and hepatocellular carcinoma cell lines. MATERIALS AND METHODS: Gastric (SNU-16, SNU-484, SNU-668) and hepatocellular (HepG2, SK-Hep-1, Hep3B) carcinoma cell lines in hollow fibers were transplanted subcutaneously and intraperitoneally into mice, which were subsequently treated with a standard anticancer agent, paclitaxel. The hollow fiber activity of paclitaxel in each cell line was compared with the xenograft activity. RESULTS: Using optimized inoculation densities and schedules, treatment with paclitaxel was effective in gastric carcinoma cell lines, SNU-16 and SNU-484, but not in SNU-668. In the hollow fiber assays, paclitaxel was effective in hepatocellular carcinoma cell lines, HepG2 and SK-Hep-1, but not in Hep3B. Consistent with the results of the hollow fiber assay, SNU-16 and SNU-484, but not SNU-668, showed tumor regression, and HepG2 and SK-Hep-1, but not Hep3B, showed effective tumor responses following treatment with paclitaxel in xenograft models. When EW7197, a novel compound, and flavopiridol were tested in SNU-16 cells under optimized conditions, the hollow fiber activity showed good correlation with the xenograft activity of each compound. CONCLUSION: Our protocols may be useful for screening candidate small molecules that may exhibit activity against stomach and liver cancers, both of which are common in Korea.


Subject(s)
Animals , Female , Humans , Mice , Appointments and Schedules , Brain , Breast , Carcinoma, Hepatocellular , Cell Line , Colon , Drug Evaluation, Preclinical , Heterografts , Korea , Liver , Liver Neoplasms , Lung , Mass Screening , Ovary , Paclitaxel , Stomach Neoplasms , Stomach
4.
Korean j. radiol ; Korean j. radiol;: 82-89, 2012.
Article in English | WPRIM | ID: wpr-28651

ABSTRACT

OBJECTIVE: To assess the relationship between apparent diffusion coefficient (ADC) values on diffusion-weighted magnetic resonance (MR) imaging and pathologic measures of a tumor using a prostate cancer xenograft model. MATERIALS AND METHODS: Eighteen athymic nude mice with 36 PC-3-induced tumors were sacrificed to obtain specimens immediately after MR imaging in order to compare the findings on MR images with those seen on pathological specimens. Using a high-field small-animal MR scanner, T1- and T2-weighted imaging and DW MR imaging was performed. Tumors were then processed for Hematoxylin and Eosin staining to evaluate tumor cellularity, intratumoral necrosis and immunostaining using antibodies directed against CD31 and vascular endothelial growth factor (VEGF) to determine the levels of microvessel density (MVD). Mean ADC values that were measured on the solid portion within each tumor were compared with tumor volume, cellularity, degree of necrosis, VEGF expression, and MVD in the corresponding section of the pathological specimen. RESULTS: Mean ADC values of the solid portion within the PC-3-induced high-grade tumors were significantly correlated with the degree of intratumoral necrosis (r = 0.63, p < 0.0001) and MVD (r = -0.44, p = 0.008) on pathologic slides. The ADC values were not significantly correlated with tumor cellularity, VEGF expression, or tumor volume in high-grade prostate cancer tissues. CONCLUSION: In the xenografted prostate cancer model, the ADC values of the solid portion of the tumors are significantly correlated with tumor necrosis and MVD of the pathologic specimens. The ADC values may be utilized as surrogate markers for the noninvasive assessment of tumor necrosis and MVD in high-grade prostate cancer.


Subject(s)
Animals , Male , Mice , Diffusion Magnetic Resonance Imaging/instrumentation , Mice, Nude , Prostatic Neoplasms/pathology , Transplantation, Heterologous , Vascular Endothelial Growth Factor A/metabolism
5.
Article in Korean | WPRIM | ID: wpr-151919

ABSTRACT

BACKGROUND/AIMS: Tetraploid cells are frequently observed in the inflamed mucosal epithelial cells of the patients with Barrett's esophagus or chronic ulcerative colitis. Polyploidy often occurs during cell fusion, abortive cell cycle, and endoreplication. Most tetraploid cells are engaged to apoptotic pathway, but some remaining stable tetraploid cells consequently cause aneuploidization and chromosomal instability. We investigated whether tetraploid cells could acquire survival advantage and hold a dominant position for natural selection. METHODS: We established tetraploid cell line (HCT116GH) from parental diploid colorectal cancer cell line (HCT116) via PEG-mediated cell fusion and compared its cell viability, cell cycle response and apoptotic fractions responded to H2O2 with diploid HCT116 and p53 suppressed HCT116/H6 cell lines. RESULTS: Using MTT assay, plating efficiency and clonogenicity, we evaluated the survival of each cell line. Tetraploid cell line HCT116GH demonstrated an 83 fold greater resistance to 100 microM H2O2 than the parental diploid HCT116, and 6 fold greater than even the p53 negative diploid HCT116/E6. Cellular sensitivity, G2/M arrests, and apoptotic proportion were observed less in response to H2O2 in HCT116GH compared with HCT116 and HCT116/E6. HCT116GH expressed lower level of p53 and p21 than diploid HCT116. CONCLUSIONS: Stable tetraploid cell lines showed enhanced viability in comparison to parental diploid cell lines. The enhanced viability observed in tetraploidization surpassed that from downregulation of p53. Frequent appearance of tetraploid cells in stressful condition can be caused by natural selection owing to their enhanced viability and may consequently contribute to cancer cell transformation.


Subject(s)
Humans , Apoptosis , Cell Division , Cell Line, Tumor , Cell Survival , Chromosomal Instability , Colonic Neoplasms/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , G2 Phase , Hydrogen Peroxide/toxicity , Oxidative Stress , Polyploidy , Tumor Suppressor Protein p53/metabolism
6.
Article in English | WPRIM | ID: wpr-97253

ABSTRACT

We report a case of combined off-pump coronary artery bypass grafting (OPCAB) and living-donor liver transplantation (LDLT). Patient was admitted to undergo liver transplantation due to Child C cirrhosis secondary to hepatitis B infection, and incidentally, his preoperative cardiac evaluation revealed silent ischemia due to the two-vessel coronary artery disease (CAD). Patient underwent OPCAB followed by LDLT. There was no perioperative cardiovascular event during the days of hospitalization. From the successful anesthetic experience of a combined OPCAB and LDLT, we cautiously suggest that a combined OPCAB and LDLT could be a surgical treatment for the patients with end-stage liver disease (ESLD) and advanced CAD.


Subject(s)
Child , Humans , Coronary Artery Bypass, Off-Pump , Coronary Artery Disease , Fibrosis , Hepatitis B , Hospitalization , Ischemia , Liver , Liver Diseases , Liver Transplantation , Living Donors , Transplants
7.
Article in Korean | WPRIM | ID: wpr-151679

ABSTRACT

Sudden hearing loss is a rare complication after general anesthesia.The variety of etiologies and the difficulty in treatment must be a challenge to anesthesiologists.In this patient who was otherwise normal in her right ear, sudden sensorineural hearing loss occurred immediately after general anesthesia.The possible causes of her sensorinerual hearing loss we supposed are the inner ear dysfunction by drilling noise or the pressure change of middle ear cavity or the microvascular circulatory deficiency related to head-rotated position.After steroid, prostaglandin injection and stellate ganglion block therapy, remarkable improvement of hearing was observed.


Subject(s)
Humans , Anesthesia, General , Ear , Ear, Inner , Ear, Middle , Hearing , Hearing Loss , Hearing Loss, Sensorineural , Mandrillus , Noise , Stellate Ganglion
8.
Article in Korean | WPRIM | ID: wpr-38585

ABSTRACT

PURPOSE: The effective treatment of an intrahepatic recurrent hepatocellular carcinoma (HCC) after a curative resection is very important in improving the prognosis after resection of HCC. The purposes of this study were to evaluate the clinicopathological characteristics and clarify the outcome of the patients after a repeat hepatectomy for a recurrent HCC. METHODS: Between March 1991 and February 2004, 16 patients underwent repeat hepatectomy for a recurrent HCC at the Yeungnam university hospital. The clinicopathological and follow-up data were retrospectively analyzed. RESULTS: There was no significant difference in the average of ICG R15 between the primary (11.2+/-1.8%) and repeat hepatectomy (18.2+/-2.8%). There were a higher proportion of minor (Couinaud's segment < or =2) resection in the repeat (93.8%) than the primary hepatectomy groups (75.0%), but the difference was not statistically significant. A significant difference was seen in the tumor size between the primary (3.6+/-0.5 cm) and repeat hepatectomy groups (2.9+/-1.9 cm). The average number of tumor in both the primary and repeat hepatectomy was equal (1.3+/-0.6). The number of cases of multicentric occurrence of HCC (12 cases) was more than that of intrahepatic metastasis of HCC (4 cases). The mean interval between the primary and repeat hepatectomy was 48.0+/-33.0 months (13~136 months). The average survival time after a primary hepatectomy was 83.6+/-36.3 months. The cumulative 1, 3, 5, and 7 year survival rates were 100, 100, 85.9, and 75.3% after a primary hepatectomy and 90, 56.5, 56.5 and 56.5% after a repeat hepatectomy, respectively. CONCLUSION: A repeat hepatectomy leads to a satisfactory outcomes in selected HCC patients.


Subject(s)
Humans , Carcinoma, Hepatocellular , Follow-Up Studies , Hepatectomy , Neoplasm Metastasis , Prognosis , Retrospective Studies , Survival Rate
9.
Article in Korean | WPRIM | ID: wpr-88533

ABSTRACT

PURPOSE: Dysregulation of apoptosis may attribute to development of cancer by abnormally prolonging cell viability with accumulation of transforming mutations. Survivin and HIAP (Human Inhibitors of Apoptosis)-1 were recently described as apoptosis inhibitors. Their pathogenic roles in gastric cancer are largely unknown. In the present study, we examined the expression of survivin and HIAP-1 in gastric cancer tissues and cell lines in order to elucidate the roles of survivin and HIAP-1 in the process of gastric carcinogenesis. MATENRIALS AND METHODS: Eight gastric cancer cell lines and five gastric cancer tissues were studied. The expression of survivin and HIAP-1 were evaluated by reverse transcription -polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot. RESULTS: Western blot and RT-PCR analysis revealed survivin and HIAP-1 expression in all gastric cancer cell lines. Increased expression of survivin and HIAP-1 were found in all cases of gastric cancer tissues compared to normal tissues by Western blot analysis. In immunohistochemical analysis tumor cells were stained with anti-survivin and anti-HIAP-1 antibodies. Cell cycle dependence of survivin expression was preserved in gastric cancer cell lines. CONCLUSION: The results indicate that increased expression of survivin and HIAP-1 genes may play an important role in gastric cancer.


Subject(s)
Antibodies , Apoptosis , Blotting, Western , Carcinogenesis , Cell Cycle , Cell Line , Cell Survival , Immunohistochemistry , Reverse Transcription , Stomach Neoplasms
10.
Exp. mol. med ; Exp. mol. med;: 361-366, 2002.
Article in English | WPRIM | ID: wpr-203700

ABSTRACT

Repetitive low dose thioacetamide (TA) treatment of hepatocytes was found to induce cells in G2 arrest. In the present study, an attempt was made to investigate alterations in expression of cell cycle regulators after G1 progression in the same repetitive low dose TA treated hepatocytes system and to define the determinators involved in G2 arrest. TA was daily administered intraperitoneally, with a dose of 50 mg/kg for 7 days. Expression levels of cyclin E and CDK2 were similar, increased at day 1 and reached a peak at day 2. And they recycled from day 3 reaching a second peak at day 5. Expression level of cyclin A was similar to p27(Kip1) and p57(Kip2) but not to CDK2 and increased to a peak level at day 2. Expression levels of cyclin B1 and cdc2 were similar although the cyclin B1 level was generally low, decreased from day 1 to basal levels at day 3 and persisted at a low level till day 7. The expression level of cyclin G1 was similar to p53 that peaked at day 3 and again at day 6 elevated over basal level. BrdU-labeled hepatocytic nuclei increased from 12 h, reached a peak at day 2, then decreased, and were not detectable from day 6. The number of PCNA-labeled nuclei increased immediately, peaked at day 2, and maintained till day 7. These results suggest that G2 arrest induced by repeated TA treatment might be p53-dependent, via activation of cyclin G1, rather than inhibition of cyclin B1- cdc2 complex, and inhibitors holding S phase progression might be p27(Kip1) and p57(Kip2).


Subject(s)
Animals , Male , Rats , Bromodeoxyuridine/metabolism , CDC2 Protein Kinase/drug effects , CDC2-CDC28 Kinases , Cell Cycle/drug effects , Cell Cycle Proteins/drug effects , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclins/drug effects , Dose-Response Relationship, Drug , G1 Phase/drug effects , Liver/drug effects , Nuclear Proteins/drug effects , Proliferating Cell Nuclear Antigen/metabolism , Tumor Suppressor Protein p53/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Rats, Sprague-Dawley , Thioacetamide/administration & dosage , Tumor Suppressor Proteins/drug effects
11.
Article in English | WPRIM | ID: wpr-179360

ABSTRACT

Fetal nucleated erythrocytes circulating in maternal blood are a potential source of fetal DNA for noninvasive prenatal genetic diagnosis. However, the estimated ratio of fetal to maternal cells is extremely small. In order to enrich these cells, we performed direct culture using a two-phase liquid system. Mononuclear cells were obtained from maternal blood samples at 8-10(+3) weeks of gestation and cultured in the first phase. After 4-5 days, the nonadherent cells were harvested and recultured with erythropoietin in the second phase for another 3-5 days. We examined cellular morphology, and counted the number of benzidine- positive cells and the percentage of glycophorin A/CD71 positive erythroid cells. We also did Kleihauer-Betke stain for Hb F, polymerase chain reaction (PCR) for SRY/DYZ1, chromosome analysis, and fluorescence in situ hybridization (FISH). The number of total erythroid cells reached about 0.1x10(6)-1.0x10(6)/mL with a purity of 84.0-97.3%. Hb F stain showed total erythroid cells of approximately 0.4x10(4)-9.8x10(4)/mL. Male DNA was detected in one case by PCR. In this case, the XY karyotype was confirmed by FISH and amniocentesis. This approach provides enriched source of fetal cells for further prenatal genetic analysis without complicated separation or sorting procedures.


Subject(s)
Female , Humans , Pregnancy , Cell Culture Techniques/methods , Cell Separation/methods , Chromosome Aberrations/diagnosis , Erythroblasts , Genetic Testing , In Situ Hybridization, Fluorescence , Karyotyping , Maternal-Fetal Exchange , /methods
13.
Article in Korean | WPRIM | ID: wpr-720633

ABSTRACT

BACKGROUND: The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic cells. There are numerous similarities between the erythroid or megakaryocytic lineages. In this study, we examined role of the region -269~-240 of gamma-globin gene promoter in fetal hemoglobin expression during either erythroid or megakaryocytic differentiation. METHODS: K562 cells were cultured and treated with differentiation inducers. Hemoglobin content was scored by benzidine staining, and hemoglobin F was stained by acid elution technique. To determine whether transcription factor binding to the gamma-globin gene promoter is critical to lineage determination, DNA-protein interaction of gamma-globin gene promoter was examined under both uninduced and induced conditions of K562 cells using gel mobility shift assay and southwestern blot analysis. RESULTS: Phorbol 12-myristate 13-acetate (PMA) induced a megakaryocytic differentiation, but suppressed erythroid differentiation. On the other hand, hydroxyurea (HU), hemin, n-butanol, and sodium butyrate (NaB) induced the expression of erythroid phenotypes. Parallel to hemoglobinization, increase in gamma-globin mRNA was observed in HU- and hemin-treated K562 cells. Gel mobility shift assay and southwestern blot analysis revealed that binding of a erythroid-specific protein (p120) to the region -269~-240 of gamma-globin gene promoter occurred with treatment of erythroid differentiation inducers and did not occur with treatment of PMA. CONCLUSION: These results suggest that erythroid differentiation inducers may act via DNA- protein interaction at the gamma-globin gene promoter region to induce erythroid differentiation.


Subject(s)
1-Butanol , Blotting, Southwestern , Butyric Acid , Cell Line , Electrophoretic Mobility Shift Assay , Fetal Hemoglobin , gamma-Globins , Hand , Hemin , Hydroxyurea , K562 Cells , Leukemia, Erythroblastic, Acute , Phenotype , Promoter Regions, Genetic , RNA, Messenger , Transcription Factors
14.
Article in Korean | WPRIM | ID: wpr-720905

ABSTRACT

We report a Korean family in which the interaction of a triplicated alpha-globin locus and a heterozygous beta-thalassemia gives rise to a clinical phenotype of thalassemia intermedia. The propositus, a 36year-old woman, was evaluated because of moderately severe chronic anemia. Molecular analysis revealed heterozygosity for a single beta-thalassemia mutation, IVSII-1 (G->A). Additionally, she was found to have co-inherited a triplicated alpha-globin gene (alphaalpha/alphaalphaalphaanti3.7). In contrast, her brother heterozygous for the same triplicated alpha-locus and beta-thalassemia was clinically normal, suggesting that the delicate balance between alpha- and beta-chains is controlled by other currently not identified factors. Thalassemia intermedia due to co-inheritance of alphaalpha/alphaalphaalphaanti3.7 and IVSII-1 (G->A) was rare, and in Korea, this patient is the first case of thalassemia intermedia attributable to this combined abnormalities.


Subject(s)
Female , Humans , alpha-Globins , Anemia , beta-Thalassemia , Korea , Phenotype , Siblings , Thalassemia
15.
Article in Korean | WPRIM | ID: wpr-96293

ABSTRACT

The present study has been performed to develop a PCR technology to identify human immunoglobulin(Ig) allotypes with restriction fragment length polymorphism(RFLP) using a probe. Genomic DNA were ampilified with PCR tecnology using primers from peripheral blood lymphocytes of 10 periodontal patiens, whose Ig allotypes have been pre-determined by serological tecnique using heagglutination technique. The result indicated that the RFLP patterns could successfully differentiate the Ig allotypes, which suggests that this technology can be developed as a tool useful for population genetics studies.


Subject(s)
Humans , DNA , Genetics, Population , Immunoglobulin Allotypes , Immunoglobulins , Lymphocytes , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length
16.
Article in Korean | WPRIM | ID: wpr-68234

ABSTRACT

BACKGROUND: Meicillin-resistant Staphylococcus aureus(MRSA) is a common cause of nosocomial infections worldwide. Identification of strains by molecular typing facilitates epidemiological studies and improves disease control This study was performed to determine the usefulness of mecA-associated hypervariable region(HVR) polymerase chain reaction (PCR) and random amplified polymorphic DNA(RAPD) analysis in the investigation of a nosocomial MRSA infections. METHODS: Methicillin-resistance was identified by NCCLS disk diffusion method using the oxacillin disk. And PCR was done for detection of mecA gene. Antimicrobial susceptibility test, HVR-PCR and RAPD using 3 primers were performed for epidemiological analysis on isolates of MRSA. RESULTS: During the period from 1997 Dec. to 1998 May, 120 strains of S. aureus were isolated from clinical specimens. Among them, 78 strains were MRSA, and 72 strains were mecA positive. The strains of mecA positive MRSA were classified into four types by antibiogram, six genotypes by HVR-PCR, and 29 groups by RAPD using three primers. The combination of HVR genotypes and RAPD analysis showed 43 different types in 72 mecA positive MRSA isolates The five strains which were repeatedly isolated from the same patients showed the same HVR genotypes and RAPD analysis. CONCLUSIONS: Antibiogram, HVR-PCR, and RAPD could classify MRSA isolates into only 4-6 types, respectively, but combination of these methods could improve the typability. And combination of results of RAPD analysis using three primers were better than that using one primer in epidemiological studies of MRSA because of same reasons. It can be concluded that molecular typing of MRSA using HVR-PCR and RAPD assay is useful in epidemiolgical investigation of nosocomial infections caused by MRSA, because of its simplicity and reproducibility.


Subject(s)
Humans , Cross Infection , Diffusion , Epidemiologic Studies , Genotype , Methicillin Resistance , Methicillin-Resistant Staphylococcus aureus , Microbial Sensitivity Tests , Molecular Typing , Oxacillin , Polymerase Chain Reaction , Staphylococcus
17.
Article in Korean | WPRIM | ID: wpr-74750

ABSTRACT

BACKGROUND: Although renal cell carcinoma (RCC) is the most common malignancy originated from kidney in adults, pathogenesis of RCC remains unknown. The purpose of this work is to find tumor suppressor gene in RCC. METHODS: The arbitrarily primed polymerase chain reaction (AP-PCR) has been used to detect somatic genetic alterations in RCC. DNA fingerprints generated by single arbitrary primers were compared between normal and tumor tissues of the same individuals. AP-PCR bands showing decreased intensities in tumor tissue DNA, relative to normal, have been cloned after reamplification with the same arbitrary primer. We have performed Southern blot hybridization and DNA sequencing. RESULTS: For a given primer, at least 5 differences in band patterns between normal and tumor tissues were observed and band C was deleted in tumor tissues of clear cell type RCC. We found this band was split into 3 bands. Because band C2 was consistantly deleted in tumor tissue, we decided to clone and characterize this fragment. Partial DNA sequences of this fragment showed no homology with other genes by BLAST search. Southern blot analysis showed this fragment was deleted in 2 cases of clear cell type and 1 case of mixed cell type RCC. CONCLUSIONS: These results suggest that fragment C2 might be a candidate for novel tumor suppressor gene and loss of this fragment might be necessary for malignant development to clear cell type RCC. Further characterization of this fragment is expected to give us useful informations about RCC tumorigenesis.


Subject(s)
Adult , Humans , Base Sequence , Blotting, Southern , Carcinogenesis , Carcinoma, Renal Cell , Clone Cells , DNA , DNA Fingerprinting , Genes, Tumor Suppressor , Kidney , Polymerase Chain Reaction , Sequence Analysis, DNA
18.
Exp. mol. med ; Exp. mol. med;: 214-220, 1998.
Article in English | WPRIM | ID: wpr-159767

ABSTRACT

Human promyelocytic leukemia cells (HL-60) have been used as a model system in which to study the effects of protein phosphatase inhibitors on NADPH-oxidase activation. Since O2- is generated by NADPH-oxidase, we examined the effect of calyculin A pretreatment on oxidase activation in response to various agonists. When Me2SO-differentiated HL-60 cells were treated with calyculin A prior to the addition of phorbol 12-myristate 13-acetate (PMA), O2- production was inhibited; however, calyculin A enhanced O2- production by N-formyl-methionyl-leucyl-phenylalanine (FMLP). The decreased O2- production seen with calyculin A pretreatment followed by PMA may be due to diminished translocation of the p47-phox and p67-phox, cytosolic components of the oxidase, and inhibition of arachidonic acid release. Interestingly calyculin A pretreatment followed by either agonist significantly enhanced mitogen-activated-protein kinase (MAPK) activity. The differential effects of pretreatment with calyculin A on subsequent oxidase stimulation elicited by FMLP or PMA provide further evidence for substantial heterogeneity in the activation of the respiratory burst.


Subject(s)
Humans , Arachidonic Acid/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Differentiation , Dimethyl Sulfoxide/pharmacology , Enzyme Inhibitors/pharmacology , HL-60 Cells , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , NADPH Oxidases/metabolism , Neutrophils/metabolism , Neutrophils/drug effects , Oxazoles/pharmacology , Oxygen/metabolism , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoproteins/immunology , Signal Transduction , Tetradecanoylphorbol Acetate/pharmacology , Time Factors
19.
Exp. mol. med ; Exp. mol. med;: 205-213, 1998.
Article in English | WPRIM | ID: wpr-159768

ABSTRACT

Since there have been very few studies on nucleolar signaling, an attempt was made to establish nucleolar signal pathways which link the cell membrane to the nucleolus for the transfer of extracellular signals. Two pathways were studied. One was the G alpha s mediated cAMP pathway where two signal molecules were yielded, including RII and protein kinase A. The other was the G alpha q mediated DAG/IP3 pathway which yields two signals including protein kinase C and IP3/Ca2+. By the studying isolated nucleoli from resting liver, regenerating liver or weak carcinogen thioacetamide treated liver, it was possible to detect protein kinase A (PKA), protein kinase C (PKC) and RII subunits. In addition, CK2 was detected. It was found that external signals transmitted through G protein coupled receptors could reach into the nucleolus and that physical translocation of signal molecules was an integral step involved in membrane-nucleolus linked pathways. When an in vitro assay of the above signal molecules was carried out using [gamma-32P]-ATP, most kinase dependent phosphorylation was via the major CK2 (more than 95%). Therefore, it is suggested that the major CK2 dependent pathway is involved in 'house keeping' for nucleolar integrity and the minor pathways, dependent on PKA, PKC and others, are involved in subtle regulatory mechanisms such as 'extra-house-keeping' activities by nucleolar chromosomal remodeling.


Subject(s)
Male , Rats , Animals , Blotting, Western , Cell Membrane/metabolism , Cell Nucleolus/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , GTP-Binding Proteins/metabolism , Immunoblotting , Liver/metabolism , Liver Neoplasms, Experimental , Models, Biological , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Kinase C/metabolism , Protein Serine-Threonine Kinases/metabolism , Rats, Sprague-Dawley , Second Messenger Systems , Signal Transduction , Thioacetamide/pharmacology , Time Factors
20.
Article in Korean | WPRIM | ID: wpr-76345

ABSTRACT

BACKGROUND: Recently, a great deal of interest has been focused on the use of hydroxyurea and hemin that may augment Hb F levels in patients with hemoglobinopathies and thalassemia, although the molecular mechanism of those chemicals remains unclear. In this study, we examined the effects of hydroxyurea and hemin on human adult peripheral and cord blood erythroid cells grown in a two-phase liquid culture system. METHODS: Four adult peripheral and four cord blood cells were cultured in two-phase liquid culture, and were treated with hydroxyurea or hemin. We counted isolated erythroid cells by acid benzidine and glycophorin A stains. To determine whether transcription factor binding to the promoter is critical, we also examined the promoter region of gamma globin gene both under uninduced and hydroxyurea or hemin induced conditions using gel mobility shift assay and southwestern blot analysis. RESULTS: When added together with erythropoietin, hydroxyurea led to significant increase in the percentage of erythroid cells in cord blood. In contrast, hemin greatly accelerated hemoglobin accumulation in adult erythroid progenitor cells. At -230 and -264 regions of gamma globin gene promoter, different protein binding patterns were observed in uninduced and hydroxyurea or hemin induced conditions between adult and cord blood. CONCLUSIONS: These results suggest that hydroxyurea and hemin may act via alteration in DNA-protein interactions to induce gamma globin gene expression. In addition, we can conclude that different transcription factors may be involved in the gamma globin induction process between the adult and cord blood erythroid cells.


Subject(s)
Adult , Humans , Blotting, Southwestern , Coloring Agents , Electrophoretic Mobility Shift Assay , Erythroid Cells , Erythroid Precursor Cells , Erythropoietin , Fetal Blood , gamma-Globins , Gene Expression , Glycophorins , Hemin , Hemoglobinopathies , Hydroxyurea , Promoter Regions, Genetic , Protein Binding , Thalassemia , Transcription Factors
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